Sample carrier and method for microscopic examination of biological samples

ABSTRACT

A sample carrier for microscopic examination of biological samples includes a base body and a filter membrane. The base body has at least one recess in which the filter membrane is disposed. The filter membrane makes an essentially flush closure with the surface of the sample carrier. The sample carrier has a circular shape.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application hereby claims priority under 35 U.S.C. §119 toGerman patent application number DE 10 2011 083 215.7 filed Sep. 22,2011, the entire contents of which are hereby incorporated herein byreference.

BACKGROUND

1. Field

Example embodiments relate to sample carriers for microscopicexamination of biological samples, and methods for detectingfluorescence-marked objects in a biological sample.

2. Description of Related Art

The separation of particles, such as cells, bacteria or the like frombiological fluids, such as blood or urine, is increasingly undertakennowadays using microfiltration. This makes use of the fact that theparticles sought (e.g., tumor cells circulating in blood) aresignificantly larger than most of the other particles or cells containedin the biological fluid. As a result, the particles separated in thisway by filtration can be colored with the usual cytological orimmunochemical methods and can subsequently be viewed, identified anddocumented under a microscope.

Thin polymer films having a porosity of the desired order of magnitudeare mostly used for microfiltration. After filtration these microfiltersare transferred onto an object carrier so that they are able to beviewed in the microscope.

This step is time-consuming and also prone to the risk of incorrecttransfer or destruction of the membrane.

A further problem in analysis of particles obtained by microfiltrationlies in the large filter surface on which the particles must be searchedfor in the microscope. In many applications the number of particlessought is so small that the entire filter surface must be evaluated inorder to obtain usable and reliable results. Since the particles soughtare small relative to the filter surface, a large number of greatlyenlarged microscope images of the filter surface must be made in orderto cover the entire filter surface, but not overlook any of theparticles sought. This too is very time-consuming and labor-intensive.

A microfilter is known from DE 10 2010 001 322 A1, which is integrateddirectly into an object carrier for microscopy. This enables the complexstep of transferring the filter to the object carrier to be dispensedwith. Even with these types of filter devices, however, the coverage ofthe entire filter surface with microphotographs is still extremelytime-consuming and complex.

SUMMARY

Example embodiments provide sample carriers and methods with which morerapid detection of marked particles in biological samples is possible.

At least one example embodiment provides a sample carrier formicroscopic examination of biological samples. The sample carriercomprises a base body with at least one recess in which a filtermembrane is disposed. The filter membrane essentially makes a flushclosure with a surface of the sample carrier. In this case, the samplecarrier may be embodied as a circular carrier. The round shape of thesample carrier makes it possible to carry out an especially rapidmicroscopic sampling of the filter surface. In contrast with samplecarriers having integrated microfilters known from the prior art, such asample carrier can be accommodated rotating under a fluorescencemicroscope or the like, so that the sampling of the entire filtersurface can be carried out more rapidly than with an object carrieraccommodated on a conventional cross table. This merely requires thesample carrier to be set into rotation, wherein a relative movement in aradial direction between sample carrier and microscope is sufficient tocapture the entire surface of the sample carrier microscopically. Thisis also relatively simple mechanically.

According to at least some example embodiments, the sample carrier has acentral, circular through-opening. A spigot of a corresponding drivespindle can engage in this opening in order to make the sample carrierrotate for analysis.

This through-opening may have a diameter of about 15 mm and an edgeheight of about 1.2 mm. This corresponds to the compact disk (CD),digital video disc (DVD) or Blu-ray standard, so that apparatuses forhandling such a sample carrier can be manufactured simply and at lowercost from components which are produced in large volumes and are thuscheaper. Other dimensions or adaptation to other standards are of coursealso possible.

In at least one example embodiment, the at least one recess, as well asthe corresponding filter membrane, is circular. This makes possible anespecially simple homogeneous application of the fluid to be filtered tothe filter membrane, which can for example be done easily by pipettingthe fluid into the center of the circular membrane.

As an alternative, the at least one recess as well as the assignedfilter membrane can also form a ring concentric to the sample carrier.On the other hand this simplifies scanning of the membrane surface onthe rotating sample carrier since the membrane surface can be capturedcontinuously and without interruption during a single rotation of thesample carrier.

The at least one recess may have an outlet channel for the filtrate.This makes possible a residue-free observation of the cells or otherparticles remaining on the filter surface as retentate.

In at least one other example embodiment, a support element forsupporting the filter membrane may be disposed in the at least onerecess. This makes it possible to use especially thin and fragile filtermembranes which, because of the support element, can easily withstandthe mechanical stress when the sample is applied, during the filtrationand during the rotational sampling.

The filter membrane may have a maximum pore size of about 20 μm, but mayalso have a pore size between about 5 μm and 20 μm, inclusive. Such afilter is especially suitable for separation of suspended tumor cells inblood, since a large part of the leukocytes, erythrocytes and othercellular blood components can easily pass through this type of filterwhile the considerably larger tumor cells will be held back on thefilter surface.

In order to simplify microscopy, the base body may be embodied fromtransparent material, such as glass, polycarbonate or the like.

At least one other example embodiment provides a method for detectingfluorescence-marked objects, such as cells, in a biological sample. Forthis purpose the sample is initially applied to a circular samplecarrier with at least one filter membrane and is filtered through thisfilter membrane. The pore size of the filter membrane in this case isselected such that the objects to be detected will be held back, whileother particles contained in the sample can pass through the filter. Tosimplify the detection and to be able to distinguish the objectsactually sought from other objects also present in the retentate, theretentate subsequently remaining on the filter membrane is treated withat least one fluorescence marker. Immunologically-coupled fluorescencemarkers are especially useful for this purpose, which for example bindthemselves specifically to surface structures of the sought cells.

After the marking of the sought objects the sample carrier isaccommodated rotatably in a holder. The holder and thereby the samplecarrier are made to rotate and the sample carrier is sampled with alaser, the frequency of which corresponds to the excitation frequency ofan assigned fluorescence marker. At the same time, fluorescence eventsare detected with a photodetector. High-resolution microscopy is not yetundertaken at this stage. Instead, the coordinates of the recognizedfluorescence events are first stored. This may be done by using a polarcoordinate system because of the circular shape of the sample carrier.Only after the complete sampling of the sample carrier with the laser isthe actual microscopy undertaken. To do this a high resolutionmicroscope is moved to the stored coordinates and microphotographs aretaken in each case.

Thus an especially rapid method is produced overall for detectingfluorescence-marked objects or particles or cells in biological samples.Through the filtration of the sample, which removes a majority of thedisruptive cellular or particulate components of the sample, especiallylow-noise viewing is made possible. Sensitivity and selectivity arefurther increased by the subsequent fluorescence marking. The method isalso compatible to using sampling facilities known per se for circularsample carriers, which are currently used to analyze samples brushedonto their surface.

A plurality of biological samples may be applied to a plurality offilter membranes of the sample carrier. This makes possible thesimultaneous or contemporaneous analysis of a plurality of samples, sothat the throughput of the method can be further increased.

The biological sample may be treated before and/or after the filtrationwith a lyze agent for lysis of the given, desired or predetermined celltype. For example, an ammonium chloride lysis can be carried out duringthe examination of blood samples in order to fragment the erythrocytespresent in large numbers and facilitate filtering them away.

The biological sample and/or further fluid media may be applied to thesample carrier by a robotic pipetting system. Such automation allows themethod to be carried out in an especially rapid and reliable manner.

It is expedient in such cases to dispose the sample carrier rotatably inthe pipetting system and to rotate the sample carrier to specificpositions for applying fluid media. This allows mechanically simplepipetting systems to be used and exploits the advantages of the circularsample carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

Example embodiments will be described in more detail hereinbelow byreferring to the accompanying drawing, in which:

The single FIGURE in this application shows a schematic view of anexample embodiment of a sample carrier.

It should be noted that these Figures are intended to illustrate thegeneral characteristics of methods, structure and/or materials utilizedin certain example embodiments and to supplement the written descriptionprovided below. These drawings are not, however, to scale and may notprecisely reflect the precise structural or performance characteristicsof any given embodiment, and should not be interpreted as defining orlimiting the range of values or properties encompassed by exampleembodiments. For example, the relative thicknesses and positioning ofmolecules, layers, regions and/or structural elements may be reduced orexaggerated for clarity.

DETAILED DESCRIPTION

Various example embodiments will now be described more fully withreference to the accompanying drawings in which only some exampleembodiments are shown. Specific structural and functional detailsdisclosed herein are merely representative for purposes of describingexample embodiments. The present invention, however, may be embodied inmany alternate forms and should not be construed as limited to only theexample embodiments set forth herein.

Accordingly, while example embodiments of the invention are capable ofvarious modifications and alternative forms, embodiments thereof areshown by way of example in the drawings and will herein be described indetail. It should be understood, however, that there is no intent tolimit example embodiments of the present invention to the particularforms disclosed. On the contrary, example embodiments are to cover allmodifications, equivalents, and alternatives falling within the scope ofthe invention. Like numbers refer to like elements throughout thedescription of the figures.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, these elements should notbe limited by these terms. These terms are only used to distinguish oneelement from another. For example, a first element could be termed asecond element, and, similarly, a second element could be termed a firstelement, without departing from the scope of example embodiments of thepresent invention. As used herein, the term “and/or,” includes any andall combinations of one or more of the associated listed items.

It will be understood that when an element is referred to as being“connected,” or “coupled,” to another element, it can be directlyconnected or coupled to the other element or intervening elements may bepresent. In contrast, when an element is referred to as being “directlyconnected,” or “directly coupled,” to another element, there are nointervening elements present. Other words used to describe therelationship between elements should be interpreted in a like fashion(e.g., “between,” versus “directly between,” “adjacent,” versus“directly adjacent,” etc.).

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of exampleembodiments of the invention. As used herein, the singular forms “a,”“an,” and “the,” are intended to include the plural forms as well,unless the context clearly indicates otherwise. As used herein, theterms “and/or” and “at least one of” include any and all combinations ofone or more of the associated listed items. It will be furtherunderstood that the terms “comprises,” “comprising,” “includes,” and/or“including,” when used herein, specify the presence of stated features,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,integers, steps, operations, elements, components, and/or groupsthereof.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which example embodiments belong. Itwill be further understood that terms, e.g., those defined in commonlyused dictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

Spatially relative terms, such as “beneath”, “below”, “lower”, “above”,“upper”, and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is turned over, elements described as “below” or “beneath” otherelements or features would then be oriented “above” the other elementsor features. Thus, term such as “below” can encompass both anorientation of above and below. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein are interpreted accordingly.

Referring to the FIGURE, according to at least one example embodiment, asample carrier for microscopic examination of biological samplesidentified overall by the number 10 comprises a circular base body 12made of a transparent material, such as glass, polycarbonate or thelike. The base body 12 has a central through-opening on which it can berotatably supported by, for example, suitable spindles, spigots or thelike. In one example, it is possible to design the centralthrough-opening 18 in accordance with the CD, DVD or Blu-ray standard,so that widely-used components can be employed to drive the samplecarrier 10.

The sample carrier 10 also has a plurality of recesses 14 let into thebase body 12. Each recess is spanned by a microfiltration membrane 16.Support elements are also provided in the recesses 14. The supportelements support and stabilize the microfiltration membrane 16, so thatit withstands the mechanical stresses of the application of the sample,filtration and rotational movement.

The properties of the microfiltration membrane 16 are governed by theactual analysis task for which the sample carrier 10 is to be employed.

In this example embodiment, the detection of tumor cells in the blood isto be illustrated by the sample carrier 10. The use of polymermicrofiltration membranes 16 with a pore diameter of between about 5 andabout 20 μm, inclusive, is suited for this purpose.

Since tumor cells circulating in the blood are only present in anextremely small number, it is expedient to first separate these fromother blood components or to concentrate them for analysis. For thispurpose the blood to be investigated is first applied to themicrofiltration membranes 16. If necessary, the separation of the tumorcells from other blood components can also be supported by anerythrocyte lysis, such as and ammonium chloride lysis. Afterapplication of the sample prepared in this way to the microfiltrationmembranes 16, the pore size of which allows the passage of lysatederythrocyte fragments, leukocytes and other small particulate bloodcomponents, while the microfiltration membrane 16 holds back thesignificantly larger tumor cells as retentate on its surface, the actualfiltration follows. The filtrate can in this case run out of therecesses 14 through channels not shown in any greater detail.

The sought tumor cells now remain on the membrane surface, as well as ifnecessary other blood components which may not have been filtered away.To facilitate the detection of the tumor cells present in extremelysmall numbers, a fluorescence coloring is undertaken in the next step.This can likewise be carried out on the surface of the microfiltrationmembranes 16. To this end, an immunofluorescence marker, which isspecific for surface proteins of the sought tumor cells, is applied tothe membrane surfaces by a pipette, where it binds itself specificallyto the corresponding targets. Likewise, depending on the cytological orimmunohistochemical coloring method used, further treatment steps arenecessary. Surplus markers can finally be washed away.

Like the application of the sample, these coloring steps can also beundertaken by an automatic pipetting system. In such cases it isexpedient to support the sample carrier 10 by the recess 18 rotatably inthe pipetting system, so that each point of the surface of the samplecarrier 10 can be reached by a radial translation movement of thepipetting robot as well as by rotation of the sample carrier 10, so thatthe pipetting system is simple to design.

After successful coloration or marking of the sought tumor cells on themembrane surface, the sample carrier 10 is brought into a correspondingdetection device. In this device, the sample carrier 10 is once againsupported rotatably on the through-opening 15. Using, for example, atleast one laser, the entire surface of the sample carrier 10 is sampledand simultaneously observed with a photo detector. The wavelength of theat least one laser corresponds to the excitation wavelength of thefluorescence colorant used. If fluorescence events are recognized, therespective coordinates are stored. Naturally in such cases it ispossible to also carry out a multiple fluorescence coloration (e.g., fordifferent surface proteins of different tumor types) to carry outspecific antibody fluorescence label complexes. Ideally the fluorescencecolorants of these complexes have different excitation and emissionwavelengths. The sampling is then undertaken in accordance with aplurality of lasers, wherein for each detected fluorescence event, notonly the coordinates but also the detected emission wavelength—and thusthe type of fluorescence label used—is determined.

If the entire surface of the sample carrier 10 or the entire surface ofthe microfiltration membranes 16 has been sampled in this way, then thedetected fluorescence events are observed microscopically in greaterdetail on the basis of the stored coordinates. A high-resolutionmicroscope moves in such cases to the stored coordinates and createscorresponding microphotographs. Because of the transparent nature of thebase body 12, this can initially be done in simple available light. Afluorescence excitation is also possible here in order to recognize thepresence of the sought tumor cells in the sample on the basis of thespecific and selective fluorescence marking. All known techniques offluorescence microscopy, such as confocal fluorescence microscopy, canbe employed here.

During the application of the sample and the analysis the sample carrier10 can be rotated in such cases at considerable speeds of severalhundred to several thousand rpm, so that especially rapid scanning ispossible. The separation of the detection of fluorescence events fromthe actual microscopic recording further speeds up the scanning in suchcases since the entire membrane surfaces do not have to be recordedmicrophotographically.

A further accelerated variant of the method is well suited to theroutine laboratory analysis of the large number of samples. In this caseblood samples of a number of patients are applied to the individualmicrofilter surfaces and simultaneously analyzed in the way described.

An alternative design of the recesses 14 and the assignedmicrofiltration membrane 16 is also possible. For example, the recesses14 and membranes 16 can run around the circumference of the samplecarrier 10 in the form of concentric rings, so that an interruption-freeobservation of the membrane surface is made possible during a completerotation of the sample carrier 10. Naturally, such ring structures arethen to be attached coaxially to the central through opening 18.

Overall the method illustrated is to be carried out especially quicklyin this way, but also at lower cost and/or in a highly selective andsensitive manner.

Example embodiments being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the present invention, andall such modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

What is claimed is:
 1. A sample carrier for microscopic examination of biological samples, the sample carrier comprising: a base body with at least one recess; and a filter membrane in the at least one recess, the filter membrane essentially making a flush closure with a surface of the sample carrier; wherein the sample carrier has a circular shape.
 2. The sample carrier as claimed in claim 1, wherein the sample carrier has a central, circular through-opening.
 3. The sample carrier as claimed in claim 2, wherein the through-opening has a diameter of about 15 mm and an edge height of about 1.2 mm.
 4. The sample carrier as claimed in claim 1, wherein the at least one recess and the filter membrane are circular in shape.
 5. The sample carrier as claimed in claim 1, wherein the at least one recess includes a plurality of recesses forming a ring concentric to the sample carrier.
 6. The sample carrier as claimed in claim 1, wherein the at least one recess has an outlet channel for filtrate.
 7. The sample carrier as claimed in claim 1, wherein a support element in the at least one recess supports the filter membrane.
 8. The sample carrier as claimed in claim 1, wherein the filter membrane has a pore size between about 5 μm and about 20 μm, inclusive.
 9. The sample carrier as claimed in claim 1, wherein the base body is embodied from a transparent material.
 10. A method for detecting fluorescence-marked objects in a biological sample, the method comprising: applying the biological sample to a circular sample carrier having at least one filter membrane; filtering the biological sample through the filter membrane; treating the retentate remaining on the filter membrane with at least one fluorescence marker; rotating the sample carrier and scanning the sample carrier with a laser while simultaneously detecting fluorescence events with a photodetector, the frequency of the laser corresponding to an excitation frequency of an assigned fluorescence marker; and storing, upon detection of a fluorescence event, coordinates of the fluorescence event on the sample carrier.
 11. The method as claimed in claim 10, wherein a plurality of biological samples are applied to a plurality of filter membranes of the sample carrier.
 12. The method as claimed in claim 10, wherein the biological sample is treated with a lysis agent at least one of before and after the filtration.
 13. The method as claimed in claim 10, wherein at least one of the biological sample and a further fluid media is applied to the sample carrier by a robotic pipetting system.
 14. The method as claimed in claim 13, wherein the sample carrier is disposed rotatably in the pipetting system and the sample carrier is rotated to specific positions for application of the further fluid media.
 15. The sample carrier as claimed in claim 9, wherein the transparent material is one of glass and polycarbonate.
 16. The sample carrier as claimed in claim 2, wherein the at least one recess and the filter membrane are circular in shape.
 17. The sample carrier as claimed in claim 2, wherein the at least one recess includes a plurality of recesses forming a ring concentric to the sample carrier.
 18. The method of claim 10, wherein after scanning the sample carrier with the laser, the method further includes, moving a high-resolution microscope according to the stored coordinates; and recording microphotographs of the biological sample at the stored coordinates.
 19. The method as claimed in claim 18, further comprising: treating the biological sample with a lysis agent at least one of before and after the filtration.
 20. The method as claimed in claim 18, wherein the sample carrier is disposed rotatably in the pipetting system and the sample carrier is rotated to specific positions for application of fluid media. 